The variable actions of opioid drugs in vivo are generally attributed to a combination of factors including receptor selectivity and efficacy for signaling, pharmacokinetics, metabolism, and ability to reach target receptors. More recently, opioid drugs have been postulated to display differential selectivity in the activation of opioid receptor homodimers/heterodimers, and in differential triggering of adaptive responses such as internalization. Here we propose that aspects of drug action may be attributed to selective opioid receptor-mediated signaling, and that different ligands, including the plethora of endogenous opioid peptides, may differentially trigger various signaling pathways via the same receptor, a phenomenon we term ligand-directed signaling. This issue will be addressed in Aim l using cell lines transfected with opioid receptors and measurement of pathways predominantly regulated by the alpha or the beta/gamma subunits of G proteins. Our preliminary data suggests that in vitro, inhibition of adenylate cyclase and activation of both MAPK and Akt can be regulated independently by different ligands following opioid receptor activation. Additionally, in mouse brain following acute morphine treatment, Akt activation appears to occur in mu expressing cells whereas MAPK activation occurs in different cells. Thus a related theme of Component II, Aims 2 through 4, will address the in vivo signaling pathways triggered by opioid ligands. We will determine why activation of the Akt pathway, but not the MAPK pathway, is specific to mu-expressing cells and whether the MAPK-activated cells are excitatory or inhibitory neurons. We will also compare the effects of acute and chronic treatment with various opioid agonists and antagonists in selectively activating these kinase pathways. These experiments will be performed in brain and spinal cord from both wildtype mice and those lacking components of the endogenous opioid or ORL-1 systems. This research could ultimately provide some insights into the role of specific signaling pathways on selective behaviors, such as analgesia, reward, sensitization or tolerance exhibited by opioid drugs.